The human cytomegalovirus (HCMV) is a significant public health concern in the United States. Most important are the effects of the virus on developing fetuses and immunocompromised individuals where it causes a variety of pathological conditions ranging in severity from mild to life-threatening. Since HCMV is present in a persistent or latent form in 50-90% of the world?s adult population, the identification of viral gene products that contribute to viral trafficking, persistence, and horizontal transmission is an intense and important area of investigation. Interestingly, HCMV encodes 4 genes that are homologous to cellular G-protein coupled receptors (GPCRs). The HCMV GPCRs are not essential for viral replication in vitro, however their homology to cellular GPCRs suggests that they may profoundly affect cellular physiology to ensure replication of the viruses in organs important for pathogenesis. The strict species specificity of HCMV has precluded an analysis of the function of HCMV GPCRs in vivo. However, as the biology of the related murine cytomegalovirus (MCMV) is similar to that of HCMV, the murine virus has served as a useful model for studying how the vGPCRs affect cytomegalovirus pathogenesis in vivo. Interestingly, the MCMV encoded M33 GPCR is essential for replication within the salivary gland of mice, suggesting that M33 may have a direct impact on persistence or horizontal transmission of virus. Based on our preliminary data, we hypothesize that human and murine CMV encoded vGPCRs activate similar Gaq-dependent signaling pathways to alter salivary acinar cell physiology and facilitate efficient amplification within the salivary epithelium. The proposed studies are highly significant as little is known about the viral and cellular properties that facilitate viral amplification within the gland and promote movement of virus into the saliva. In aim 1, we will use knockout and pharmacological approaches to test the hypothesis that Gaq/Ga11 is the proximal signaling pathway used by M33 for MCMV growth within the salivary gland in vivo. In aim 2, we will use novel animal models to examine cellular gene expression in salivary acinar epithelial cells infected in vivo and determine how MCMV vGPCR-induced changes provide physiological adaptations critical for optimal cytomegalovirus growth in the gland. In aim 3, we will use primary salisphere-derived organoids and test whether salivary epithelial cells similarly require CMV vGPCR activity to promote viral growth in vitro and in vivo. In aim 4 we will examine signaling properties of the HCMV and MCMV vGPCRs in the organoid systems. The innovative experiments proposed in this application will lead to important insight into the function of cytomegalovirus vGPCRs in vivo and define mechanisms by which cytomegaloviruses persist and gain access to fluids important for horizontal transmission. Defining the essential roles for cytomegalovirus vGPCRs in promoting salivary gland replication and spread could ultimately lead to the development of unique antivirals designed to prevent cytomegalovirus transmission via saliva.